1. Field of the Invention
This invention relates to a DNA polymerase suitable for DNA amplification. More specifically, this invention related to a bacteriophage Φ15 DNA polymerase having strand-displacement activity.
2. Description of Related Art
DNA sequencing involves the generation of four populations of single-stranded DNA fragments, having one defined terminus and one variable terminus. The variable terminus always terminates at a specific given nucleotide base (either guanine (G), adenine (A), thymine (T), or cytosine (C)). The four different sets of fragments are each separated on the basis of their length, on a high resolution polyacrylamide gel; each band on the gel corresponds colinearly to a specific nucleotide in the DNA sequence, thus identifying the positions in the sequence of the given nucleotide base.
Generally there are two methods of DNA sequencing. One method (Maxam and Gilbert sequencing) involves the chemical degradation of isolated DNA fragments, each labeled with a single radiolabel at its defined terminus, each reaction yielding a limited cleavage specifically at one or more of the four bases (G, A, T or C). This method generally requires no DNA polymerase and has lost popularity as the other, dideoxy sequencing method has become improved in many ways over the past 25 years.
The second method (dideoxy sequencing) involves the enzymatic synthesis of a DNA strand. Four separate syntheses are run, each reaction being caused to terminate at a specific base (G, A, T or C) via incorporation of the appropriate chain terminating dideoxynucleotide. The latter method is preferred since the DNA fragments are uniformly labelled (instead of end labelled) and thus the larger DNA fragments contain increasingly more radioactivity. Further, 35S-labelled nucleotides can be used in place of 32P-labelled nucleotides, resulting in sharper definition; and the reaction products are simple to interpret since each lane corresponds only to either G, A, T or C. The enzymes used for most dideoxy sequencing is the Escherichia coli DNA-polymerase I large fragment (“Klenow”), AMV reverse transcriptase, and T7 DNA polymerase (Tabor et al., U.S. Pat. No. 4,795,699). The T7 DNA polymerase used for sequencing is said to be advantageous over other DNA polymerases because it is processive, has no associated exonuclease activity, does not discriminate against nucleotide analog incorporation, and can utilize small oligonucleotides as primers. These properties are said to make the polymerase ideal for DNA sequencing. Id.
A means of amplifying target DNA molecules is of value because such amplified DNA is frequently used in subsequent analysis methods including DNA sequencing, cloning, mapping, genotyping, generation of probes, and diagnostic testing.
There are several established methods that permit amplification of nucleic acids. Most of these methods were designed around the amplification of selected or specific DNA targets using specific probes or primers. Examples include the polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), and amplification with Q.βreplicase (Birkenmeyer and Mushahwar, J. Virological Methods, 35:117-126 (1991); Landegren, Trends Genetics, 9:199-202 (1993)).
In addition, several methods have been employed to amplify circular DNA molecules such as plasmids or DNA from bacteriophage such as M13. One (cloning) is simply the propagation of these molecules in suitable host strains of E. coli, followed by isolation of the DNA by well-established protocols (Sambrook, J., Fritsch, E. F., and Maniatis, T. Molecular Cloning, A Laboratory Manual, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). PCR has also been a frequently used method to amplify defined sequences in DNA targets such as plasmids or DNA from bacteriophage such as M13 (PCR Protocols, 1990, Ed. M. A. Innis, D. H. Gelfand, J. J. Sninsky, Academic Press, San Diego.) Some of these methods suffer from being laborious, expensive, time-consuming, inefficient, and lacking in sensitivity.
As an improvement on these methods, linear rolling circle amplification (LRCA) uses a primer annealed to a circular target DNA molecule and DNA polymerase is added. The amplification target circle (ATC) acts as a template on which new DNA is made, with the polymerase extending the primer as a continuous strand. Once the polymerase has completed a full circuit of the template, it displaces the product strand and continues copying the template forming a series of repeats of the sequence complementary to the circle. This process can generate hundreds or even thousands of copies of the template during several hours of reaction, with the number of copies increasing linearly (not accelerating) with time. An improvement on LRCA is exponential RCA (ERCA). To achieve exponential or accelerating amplification, an additional primer that anneals to the replicated complementary strand is provided. This creates new centers of amplification on the product strand, thereby providing accelerating, exponential kinetics and increased amplification. Exponential rolling circle amplification (ERCA) employs a cascade of strand displacement reactions, also referred to as HRCA (Lizardi, P. M. et al. Nature Genetics, 19, 225-231 (1998)). However, ERCA is limited to the use of just a single primer annealed to the circular DNA target molecule, and a single primer for the product strand. Another limitation is that one needs to know a specific DNA sequence within the circular target sequence in order to make the primers. Furthermore, the circular DNA target molecule must be a circle that is nicked or at least partially single-stranded.
Another method (referred to herein as Multiply-Primed Rolling Circle Amplification—MPRCA) that avoids some of these disadvantages employs multiple specific primers to perform rolling circle amplification by using multiple primers for the amplification of individual target circles. This has the advantage of more rapid synthesis with multiple growing points on each circular template molecule. Amplification can be linear if all the primers anneal only to the circular target strand, or exponential if primers are provided for both strands.
The methods provide a mechanism for “in vitro cloning”, i.e. without the need for cloning into an organism, of known or unknown target DNAs enclosed in circles. A padlock probe may be used to copy the target sequence into a circle by the gap fill-in method (Lizardi, P. M. et al. Nature Genetics, 19, 225-231 (1998)). Alternatively, target sequences can be copied or inserted into circular ssDNA or dsDNA by many other commonly used methods. The RCA amplification overcomes the need to generate amplified yields of the DNA by cloning in organisms.
While specific target amplification is widely used, general amplification of all the sequences in a target sample can also be useful. This can be achieved with a form of MPRCA in which a collection of primers of arbitrary or random sequence is used. In this way, amplification of a circular target DNA molecule of unknown sequence can be achieved. Another advantage is that the amplification of single-stranded or double-stranded circular target DNA molecules may be carried out isothermally and/or at ambient temperatures. Other advantages include being highly useful in new applications of rolling circle amplification, low cost, sensitivity to low concentration of target circle, flexibility, especially in the use of detection reagents, and low risk of contamination (U.S. Pat. No. 6,323,009; Genomics 2002 Vol. 80, No. 6, 691-8; Biotechniques 2002 June Suppl. 44-47; Genome Research, 2001 Vol. 11 No. 6, 1095-9).
In some embodiments, procedures are employed that improve on the yield of amplified product DNA by using multiple primers that are resistant to degradation by exonuclease activity that may be present in the reaction. This has the advantage of permitting the primers to persist in reactions that contain an exonuclease activity and that may be carried out for long incubation periods. The persistence of primers allows new priming events to occur for the entire incubation time of the reaction, which is one of the hallmarks of ERCA and has the advantage of increasing the yield of amplified DNA.
Random primer RCA also has the benefit of generating double stranded products. This is because the linear ssDNA products generated by copying of the circular template will themselves be converted to duplex form by random priming of DNA synthesis. Double stranded DNA product is advantageous in allowing for DNA sequencing of either strand and for restriction endonuclease digestion and other methods used in cloning, labeling, and detection.
Methods have published for whole genome amplification using degenerate primers (Cheung, V. G. and Nelson, S. F. Proc. Natl. Acad. Sci. USA, 93, 14676-14679 (1996)) and random primers (Zhang, L. et al., Proc. Natl. Acad. Sci. USA, 89, 5847-5851 (1992)) where a subset of a complex mixture of targets such as genomic DNA is amplified. Reduction of complexity is an objective of these methods.
It has been found, however, that the use of the DNA polymerase from bacteriophage Φ29 (PHI29) along with random sequence hexamer primers is particularly advantageous for general amplification of circular DNA sequences by exponential MPRCA mechanism. In addition, it has also been found that this combination is also effective in amplification of linear DNA molecules in a process termed MDA for Multiple Displacement Amplification. It is capable of amplification of the 50 kb chromosome of bacteriophage λ, or of larger linear molecules such as isolated human chromosomal DNA. This method makes use of the unusual properties of Φ29 DNA polymerase, namely high processivity and ability to displace a strand annealed to the template strand during synthesis—so called strand-displacement activity (Proc Natl Acad Sci USA. 2005 May 3;102(18):6407-12). Most DNA polymerases slow or stop when they encounter a strand annealed to the template but Φ29 DNA polymerase has the same synthesis rate on single-stranded DNA as when it must displace a strand from a double-stranded template. Also, most DNA polymerases remain bound to a template strand long enough to polymerize only a few nucleotides up to about 1000 nucleotides. Synthesis at that particular site then stops as the polymerase un-binds, and synthesis will only resume when another molecule of polymerase binds later. In contrast, Φ29 DNA polymerase appears to bind long enough to polymerize many thousands of nucleotides without stopping, extending synthesis over wide stretches of template sequence. Together, these unique properties of Φ29 DNA polymerase make it particularly effective for MDA and MPRCA using random-sequence primers such as hexamers.
Experiments using a variety of DNA polymerases have revealed that many of these enzymes do not support amplification by MDA. This is presumably because these enzymes will slow or stop whenever they encounter a double-stranded region of the template DNA such as a region already copied by synthesis on single-stranded template. Thus, synthesis stops when all of the single-stranded DNA in the sample is converted to double stranded DNA. This limits amplification to 2-fold at most while the Φ29 DNA polymerase readily amplifies DNA one million-fold or more.
New polymerases are desirable. This need is addressed in greater detail below.